The intracellular level of cholesterol and cholesteryl esters controls the level of the microsomal enzyme 3-hydroxy-3-methylglutaryl Coenzyme A (HMG-CoA) reductase and thereby regulates the rate of cholesterol synthesis. The long-term goal of these investigations is to elucidate the molecular and biochemical mechanisms through which cholesterol controls the level of HMG-CoA reductase. Our approach is to establish the site at which exogenous cholesterol suppresses HMG-CoA reductase synthesis in established cell cultures of animal and human origin. Normal liver cell cultures adapted for growth in chemically defined medium and regulatory variants defective in regulation by oxygenated sterols or cholesterol are employed in these investigations. The site of cholesterol action will be established through immunochemical studies of HMG-CoA reductase synthesis. The effects of cholesterol on the level of HMG-CoA reductase specific messenger RNA, on the efficiency of translation of HMG-CoA reductase mRNA in vivo and on the integration of newly synthesized HMG-CoA reductase into the endoplasmic reticulum will be assessed. The HMG-CoA reductase mRNA content of RNA preparations will be determined by indirect immunoprecipitation of the reductase synthesized in a cell free protein synthesizing system derived from nuclease treated rabbit reticulocyte lysate. The location, number and size of HMG-CoA reductase synthesizing polyribosomes will be determined in iodinated antibody binding studies and by mRNA assay and the possible occurence of translation control and a hydrophobic "signal peptide" on newly synthesized reductase will be examined. The regulatory variants produced can serve as models for clinical syndromes in which defective cholesterol regulation of HMG-CoA reductase results in serum hypercholesterolemia and atheroschlerosis. Cholesterol regulation of HMG-CoA reductase synthesis represents a favorable system for investigation of the mechanisms governing gene expression and protein synthesis in animal and human cells.